In the field of genetic engineering, as a method allowing direct analysis of a genetic feature, analysis based on the complementarity of nucleic acid sequences has been known. In such analysis, when the amount of a target gene in a sample is small, the detection thereof generally cannot be achieved easily. Thus, it is necessary to amplify the target gene itself in advance.
Amplification of the target gene (nucleic acid amplification) chiefly is carried out by an enzymatic method utilizing a DNA polymerase. Major examples of such an enzymatic method include: a PCR method (Polymerase Chain Reaction method; U.S. Pat. No. 4,683,195 (Patent Document 1), U.S. Pat. No. 4,683,202 (Patent Document 2), and U.S. Pat. No. 4,800,159 (Patent Document 3)); and a RT-PCR method (reverse transcription PCR method; Trends in Biotechnology 10, pp. 146-152, 1992 (Non-Patent Document 1)), which is a PCR method combined with a reverse transcriptase reaction. These methods allow the target gene to be amplified from DNA or RNA by repeating a reaction including the following three stages: dissociation of a double-stranded nucleic acid serving as a template into single-stranded nucleic acids (denaturation); annealing of a primer to the single-stranded nucleic acid; and synthesis of a complementary strand from the primer (extension). In these methods, the temperature of the reaction solution needs to be adjusted to temperatures suitable for the above-described three stages, and these three steps of temperature adjustment need to be repeated.
Furthermore, European Patent Publication No. 0320308 (Patent Document 4) discloses a ligase chain reaction method (LCR method), in which a known gene sequence is amplified by performing a two-step thermal cycling reaction (reaction repeating heating and cooling) using a heat-resistant DNA ligase. However, in the methods described above, it is necessary to use an expensive thermal cycler that can achieve strict temperature control over time over a wide temperature range. Besides, these reactions are carried out under two to three kinds of temperature conditions. Thus, time for adjustment to each reaction temperature is required, and the time required increases in keeping with the number of cycles.
In order to solve the above-described problems, there have been developed nucleic acid amplification methods that can be carried out under isothermal conditions. Examples of such methods include: an SDA (strand displacement amplification) method described in JP 7(1995)-114718 B (Patent Document 5); a 3SR (self-sustained sequence replication) method; an NASBA (nucleic acid sequence based amplification) method described in Japanese Patent No. 2650159 (Patent Document 6); a TMA (transcription-mediated amplification) method; a Q beta replicase method described in Japanese Patent No. 2710159 (Patent Document 7); various kinds of improved SDA methods described in U.S. Pat. No. 5,824,517 (Patent Document 8), WO 99/09211 (Patent Document 9), and WO 95/25180 (Patent Document 10); a LAMP (Loop-Mediated Isothermal Amplification) method described in WO 00/28082 (Patent Document 11); an ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method described in WO 02/16639 (Patent Document 12); and a SmartAmp2 method described in Japanese Patent No. 3897805 (Patent Document 13). In these isothermal nucleic acid amplification methods, reactions at all the stages proceed simultaneously in a reaction mixture kept at a given temperature.
Among the above-described isothermal amplification methods, the LAMP method and the SmartAmp2 method are superior in practical utility. The LAMP method is an isothermal amplification method in which two turn-back primers (TPs) and two outer primers (OPs) are essential. Thus, in the LAMP method, four kinds of primers are required, and genome recognition sites are six in total. FIG. 11 illustrates an example of the LAMP method. In FIG. 11, the two OPs are omitted, and only the two TPs are shown. As shown in FIG. 11, each of the TPs includes, on the 3′ side thereof, a sequence that hybridizes to a target nucleic acid sequence, and includes, on the 5′ side thereof, a sequence complementary to the primer-extended strand. For example, in FIG. 11, one of the TPs (on the left in FIG. 11) includes, on the 3′ side thereof, a sequence (A′) complementary to a sequence (A) of the target nucleic acid sequence, and includes, on the 5′ side thereof, a sequence (M) complementary to a sequence (M′) of the primer-extended strand. The other TP (on the right in FIG. 11) has the same structure. When the TP with the above structure hybridizes to the template sequence and an extended strand thereof is formed, the 5′ side of the TP turns back to hybridize to the extended strand. As a result, a stem-loop structure is formed on the 5′ side of the primer-extended strand. Because two TPs are used in the LAMP method, the LAMP method has a problem in that it is difficult to shorten the sequence of a region to be amplified in the target nucleic acid sequence. Moreover, because four kinds of primers are required and there are six genome recognition sites in total in the LAMP method as described above, the LAMP method also has a problem in that primer design is difficult. On the other hand, the SmartAmp2 method uses a TP and a folding primer (FP), so that the above-described problems in the LAMP method do not occur in the SmartAmp2 method. FIG. 12 illustrates an example of the SmartAmp2 method. As shown in FIG. 12, in the SmartAmp2 method, one of the primers is a TP and the other primer is a FP. As shown in FIG. 12, the FP includes, on the 3′ side thereof, a sequence (B′) complementary to a sequence (B) of a target nucleic acid sequence, and includes, on the 5′ side thereof, a folding sequence including sequences F-F′ that are complementary to each other on a single strand. Because the SmartAmp2 method uses the TP and the FP, there are three genome recognition sites. Besides, the FP does not turn back. Accordingly, the SmartAmp2 method is advantageous not only in that it achieves high amplification speed and high specificity but also in that it allows easy primer design and shortening of a region to be amplified.